Structure Opiitzation And Function Study Of Bt Gene

Swedish scientists firstly introduced Bacillus thuringiensis (Bt) crystal toxin gene into tobacco andpotato through transgenic approaches in1987. After this Landmarker event, many genetical modification(GM) crops, carrying novel traits such as insect-resistant and herbicide-tolerance, have been developed invarious plants. Bt cotton is one of most widely commercialized GM crops across the world. Following theUnited States, Chinese scientists successfully developed the Bt cotton with independent intellectual propertyright in1997. The Bt cotton has been widely and rapidly adopted by farmer in China because Bt cottondisplayed good performances such as insecticide-saving, high-yield and environment-friendly. Bt cottonaccounts for more than80%of the total planting cotton in2010. With the advantage of Bt cotton, its socialand economic benefits have been widely recognized by the governments and peoples. However, there stillhave some underlying limitations and problems remaining to be resolved in current cotton molecularbreeding: Firstly, with the massive use of Bt cotton, insect pests will quickly develop resistance to cotton withBt toxin eventually resulting in the potential environmental risks. Secondly, the dosage of Bt toxin is still lowin reproductive stage of cotton leading to Bt cotton reduce the function of insect-resistant at late stage ofcotton. Thirdly, the cotton genetic transformation methods mainly depend on tissue culture basedAgrobacterium-mediated approach and this method is strongly depended upon the cotton genotype, which isseverely restricted the cotton improvement through genetically modification. According to the aboveproblems, this paper achieved some new methods and strategies to resolve them:1) Recent report discovered that deletion of Cry1Ac α-helix domain is able to enhance the toxicityof Bt toxin. We therefore deleted the first α-helix domain, containing129bp nucleotide acids, oflaboratory storage Bt (GFM Cry1A) gene. The Bt protein truncation constructs: BtΔα was designed andmade. Furthermore, BtΔα fusing with Cpti, another protein toxin, FBCΔα was also successfullydeveloped. Bt and Bt:Cpti fusion genes were set as control. Prokaryotic expression vocter PT7: BtΔαand PT7: FBCΔα based on PET-28a vector backbone has been constructed and introduced into E.coliBL21strain. The protein BtΔα and FBCΔα have been expressed and purified for toxin activity assay.The results showed that the LC50of sensitive bollworm and resistant bollworm. For sensitive bollworm:Bt protein LC50was161.93ng/g, BtΔα protein LC50was104.08ng/g, FBC protein LC50was of148.85ng/g, FBCΔα protein LC50was100.81ng/g. For5times Bt toxin resistant bollworm: Btprotein LC50was>6400ng/g, BtΔα protein LC50was491.04ng/g, FBC protein LC50was>6400ng/g, FBCΔα protein LC50was448.41ng/g. Additionally, the plant expression vocter P35s: BtΔα, P35S:FBCΔα, P35s: Bt, P35S: Cpti, have been constructed and transformed into tobacco. Through analysisthe obtained transgenic tobacco by the PCR, ELISA, bioassay and field pick insects experimental, theresults shows that BtΔα protein, FBCΔα are able to expressed in tobacco and with insecticidal activity.2) Recent studies showed that mitochondrial targeting peptide and chloroplast targeting peptideenable to significantly increase the dosage of exogenous protein in the cells. We therefore isolated anArabidopsis ALDH gene containing mitochondrial targeting peptide nucleic acid sequences AtMTP anda cotton EPSPS gene containing chloroplast targeting peptides nucleic acid sequences GhCTP, respectively. After fused them with the Bt gene and the GFP gene,respectively, then transformed intotobacco. The GFP signal has been detected by Fluorescence microscopy in the transgenic tobacco,indicating that two signal peptide has the expected signaling targeting activity. The results suggestedthat the dosage of Bt protein can be increased double times in transgenic tobacco through fusing Btprotein with mitochondria and chloroplasts targeting peptides.3) Established flower spraying Agrobacterium method for cotton genetic transformation. Basically,spinning and collection the OD600=0.8agrobacteria containing the target constructs; re-suspension thepellet in10%sucrose and0.05%SilWet L-77solution; spraying the mixture solution on the stigma andanther of cotton unopened flower in the morning. The spraying tool is a little aerosol spray bottle. Byusing the method,42T0transgenic cotton were obtained via this approach. The transformat efficiency isabout9%. Five independent transgenic lines were selected for further analysis by Southern blot andElisa. The results suggested that the exogenous genes were integrated into cotton genome and can bestably passed down from T1generation to T3generation.In short, through exploration of optimization of Bt protein； mitochondria and chloroplastsubcellular localizated expression of Bt protein in the tobacco and improvement of genetictransformation methond in cotton, we developed an improved method for cotton genetic transfomation,increasing the dosage of Bt toxin in plant cell, optimization the Bt protein structure and subsequentlyincreasing the toxicity of Bt protein in transgenic cotton in this study.